JP2722134B2 - Cryopreservation of cultured epithelial cell sheets - Google Patents

Abstract

Disclosed is a method of cryopreserving a sheet of living, cultured epithelial cells for use as a skin wound dressing. The method ensures that the integrity of the sheet will be maintained, and it preserves a significant number of cells in a mitotically competent, physiologically healthy form. It involves use of a non cell penetrating cryoprotectant and certain freezing and thawing conditions.

Description

Description: BACKGROUND OF THE INVENTION The present invention relates to the cryopreservation and long-term storage of cultured epithelial tissue sheets having cell viability and colony-forming ability and useful as dressings for skin wounds.

The medical community has emphasized the development of a skin wound dressing that promotes the growth of new cells while preventing serum leakage due to burns, ulcers, surgical resections, etc. and preventing infection of skin wounds. Conventional dressings cannot adequately protect widespread wounds, and several alternatives have been developed. These replacement methods include a method of transplanting cut or completely thick cadaver skin or pig skin, human allograft, and human autograft. Unless immunosuppressive therapy was employed, rejection, except autologous transplantation, was in most cases unsuccessful. Furthermore, conventional autograft techniques have not been feasible for severe burns that cover a large area.

Green et al. Have developed a method of culturing epithelial cell sheets with several layers of cells to treat burns, ulcers, and other skin wounds. US Patent 4,016,036
The publication discloses a method for continuously culturing keratinocytes to produce a layered sheet of epithelium. U.S. Pat. No. 4,304,866 discloses a method for producing an implantable cell sheet by culturing keratinocytes and peeling the cell sheet from the adherent support using an enzyme such as despase. U.S. Pat. No. 4,456,687 discloses agents useful for promoting epithelial cell growth. The disclosures of these patents are incorporated herein by reference. In the culture system developed by Green et al., Epithelial cells rapidly divide on the surface of a tissue culture dish or flask, eventually forming a confluent, moderately layered, tightly bonded sheet of cells. . These confluent cultures can be published as bound cell sheets, for example, by treatment with the enzyme despase (see US Pat. No. 4,304,866). The culture sheet is
Attached to vaseline-soaked gauze or other non-adherent support, transported to the operating room while still in the culture medium, and used for patient treatment.

A wide range of burns can be covered with autograft materials prepared by these methods. However, autologous transplantation requires time for culture. While culturing cells for autologous transplantation, the wound can be protected by an allogeneic transplant, which is effective as a temporary wound dressing. The allograft material promotes the healing of chronic skin ulcers and scars on the exfoliated skin graft supplier. Cultured autologous material prepared by the method of Green et al. Is available from BioSurface Technology, Inc., Cambridge, Mass. Allograft material is available for experimental and clinical trial use.

A serious practical problem in the use of cultured epithelial cell transplantation is the inherently short shelf life. The viability and colony forming ability of the sheet are rapidly lost when leaving the medium in which the sheet is growing. This limits the time and distance that the sheet travels from the production facility to the operating room. Cell sheets are extraordinarily brittle. This cell sheet can maintain the function of growth and colony forming ability when treated on a wound surface at most within 6 to 8 hours after exfoliation with dispase. Time constraints hinder securing large cell sheets. The development of cryopreservation techniques to extend the storage time of culture sheets will enable the maintenance of large numbers of cell sheets that can be transported worldwide.

The prior art is full of descriptions of various tissue preservation methods, including cryopreservation, the use of specific cell media, and reliable packaging techniques. Cryopreservation allows a substance to be frozen and stored for long periods of time in the presence of a cryoprotectant. The drug eliminates aqueous material inside and outside the cells and prevents the formation of ice crystals. There are a number of disclosed protocols regarding the nature and amount of cryoprotectants and / or the time, course, temperature, etc. of the freezing step in an attempt to maintain cell viability after a freeze-thaw cycle. For example, U.S. Pat.
No. 4,688,387.

Preserving tissue by cryopreservation is a complex and expensive process and can produce highly variable results. However, no other method has extended the storage viability of animal tissues for a very short time, for example, more than about 8 hours. For example, Pittelkow et al. (JI
nvest. Dermatol. 4 : 410-17, 413-14, 1986). For a long time, skin banks have used frozen human skin for burns as a temporary allograft coating. However, this freeze is not very practical. Although preserved skin is metabolically active, it is not certain that epithelial cells can renew themselves (Hemibach, D., et al., Art.
ificial Dermis for Major Burns: "a Multi-Center Ra
ndomized Clinical Trial ", Ann, Surg. 313-320, Septemb
1988).

As noted by Green et al., The production of large-scale cultured epithelial autografts has facilitated permanent and extensive coverage of wounds with their own skin. Although large quantities of cultured epithelium are produced for patients, the epithelial life span is limited and presents a major problem. As a result, Canseda and Delucca have developed a method of freezing confluent sheets of cultured keratinocytes in a culture solution containing 10% glycerol (see EP 0 296 475). However, experience with this method has confirmed that the rate of recovery of the cells fluctuates and is generally very low. Furthermore, the range of incubation times was very narrow and proved impractical for large-scale production. Moreover, transplants frozen by some protocol and stored and thawed in liquid nitrogen are often cracked. Wound covering may be possible, but at the cellular level, problems remain regarding the active state of the confluent sheet.

Early studies using high molecular weight cryoprotectants have shown that polyvinylpyrrolidone (PVP) and dextran (MW 3
0-100 Kd) have been found to prevent red blood cell destruction during cryopreservation (pegg, DE, "Banking of Cells,
Tissues and Organs at Low Tempratures ", Current Tre
nds in Cryobiology, edited by A. Smith, 1970). The combination of glycerol with dextran and hydroxylethyl starch partially preserves the freezing and thawing power of sheep sperm (Schmehl et al., "The Effests of Nonpenetrating").
Cryoprotectants Added to Tesr-Yolk-Glycerol Exte
nder on the Post-Thaw Motility of Ram Spermat ozo
a "23 Cryobiol. 6: 512-17, 1986).

When trypan blue dye exclusion is performed as an indicator of viability, it has been confirmed that only hydroxylethyl starch (HES) promotes the viability of cryopreserved cells that are hematopoietic sources. However, it is only effective if the cryoprotectant is added and then slowly removed in a very time-consuming manner (Conscience, Fischer, "An Improved
Preservation Technique for Cells of Hemapoetic Ori
gin "22 Cryobiol. 5: 495-98, 1985). This viability parameter only provides information on short-term membrane stability, and provides data on tissue growth potential and long-term viability. No, the authors state that HES is ineffective at cryopreserving cells from epithelial tissue, and another study found parameters that more closely reflect long-term viability of cells. However, as determined by cell proliferation assays, cryopreservation of hematopoietic cells
Was effective (Wang, et al., Cryobiol, 24: 229-237, 1
987).

Studies using high molecular weight non-permeable (glass-forming) agents as cryoprotectants tend to concentrate on floating cells such as the red blood cells and lymphocytes described above. However, the need to maintain the integrity of the coherent sheet of cells during cryopreservation severely limits the recovery of viable cells (ie, cells capable of tissue regeneration).

It is an object of the present invention to provide a cryopreservation methodology that can preserve a live culture sheet of epithelial cells. In this methodology, the method preserves the structural integrity of the sheet to enable the formation of epithelial tissue useful for treating wounds, and also maintains the mitotic capacity of the cells of the sheet. It is.

SUMMARY OF THE INVENTION A novel method of cryopreserving a confluent sheet of cultured live epithelial cells has been discovered so that the utility of the skin as a wound dressing can be maintained. According to this method, the colony forming ability of the cells in the sheet can be maintained, that is, a significant number of the cells in the epithelial cell sheet can be mitotically separated so that healthy epithelium can be regenerated. Live cells can be stored. The method further allows the cultured epithelial sheet to be harvested when the sheet is matured and stored in a cryopreserved state for future use.

The method comprises the following steps. 1) At least
The sheet is immersed in a cryopreservation solution containing a non-cell permeable glass former and preferably a cell permeable glass former. 2)
The sheet is cooled and frozen at least below -65 ° C, more preferably below -120 ° C (glass transition temperature of water), and particularly preferably below -180 ° C, which is the long-term storage temperature. As a preferred cooling method, it is desirable to cool the liquid nitrogen in an evaporating atmosphere at about -180 to -196 ° C. 3) As the next step, at least -65 ° C
Store the sheet at approximately -180 ° C below. 4)
Thawing is performed to prepare a complete sheet of cells having a colony forming capacity of at least 35%, often 40% and often 50% or more.

The cryoprotectant solution contains the following components in superimposed percentages: About 5-20%, preferably 15%, of the non-cell penetrating glass former and about 10% of the cell penetrating glass former. Glycerol, propylene glycol, ethylene glycol dimethyl sulfoxide (DMSO) or a mixture thereof is used as a cell permeable glass forming agent. Glycerol is particularly preferred as the cell permeable glass former. Dextran or a derivative thereof, polyvinylpyrrolidone, hydroxyethyl starch, chondroitin sulfate, poly (ethylene glycol),
Alternatively, use a mixture thereof. Alkylated or etherified derivatives are used as dextran derivatives.
Glycerol is particularly preferred as the cell permeable glass former. Dextran, polyvinylpyrrolidone, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, or mixtures or derivatives thereof are used as non-cell permeable glass formers. Dextran and hydroxyl starch are particularly preferred.

In a preferred embodiment, the freezing step requires gradual cooling of the sheet to at least about -4 ° C and to -80 ° C. It is then cooled from a liquid nitrogen vapor temperature of approximately -180 ° C and to a temperature in the range of -196 ° C. Slow cooling is performed at a cooling rate of about 1 ° C./min. More preferably, the melting step requires warming the sheet from low storage temperature to -120 ° C and to -80 ° C for one to five minutes using air or other gas. That is, the heating rate is from about 20 ° C./min to about 100 ° C./min. Further, the sheet can be cultured in a water bath or raised to a temperature of about 20 ° C to about 37 ° C.

Prior to use as a skin wound dressing, the thawed sheet is free of cryoprotectants and at physiological pH, e.g., isotonic buffer, preferably Ringer's lactate, to allow cells to be stored temporarily before use. Rinse with. The epithelial cells of the sheet used in the present method are preferably cultured keratinocytes. Confluent sheets are several layers of cells thick and consist of different layers.

Other objects and features of the present invention will become apparent from the following description of the drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a cross-sectional view of a layered cultured keratinocyte sheet released from a culture flask by a despase. 1 is a representative micrograph of a layered cultured keratinocyte sheet using the method of the present invention.

FIG. 2 shows in a bar graph the effect of using a non-cell penetrant for cryoprotection. 10 cultured keratinocyte sheets
Equilibrate for 6 minutes (gly c-6) in cryopreservation culture (CPM) containing 10% glycerol and then for 30 minutes (gly c-30) or cryopreservation containing 10% glycerol containing 15% dextran (70 kd) Equilibrate for 30 minutes in culture (CPM)
Then, it was cooled to -180 ° C and stored at -180 ° C for 2-3 days.
After thawing, viability was measured by whole cell recovery (R), colony forming ability (CFE), colony forming cells (CF
The viability of C) was evaluated and measured. CFC = recovered cells × recovered cells CFE (non-frozen, non-preserved control sheet expressed as a percentage) FIG. 3 shows the effect of dextran and hespan (hydroxylethyl starch) on the survival of cryopreserved cultured epithelial grafts Is shown by a bar graph. Implants are 15% in 10% glycerol
Equilibrated in CPM containing either% Hespan, 15% Dextran (70 kd) or 15% Dextran (500 kd), cooled to -180 ° C and stored for 2 days. The viability after thawing was measured by evaluating R, CFE and CFC. Expressed as a percentage of unfrozen, non-preserved control grafts.

FIG. 4 is a bar graph showing the effect of cryoprotective medium before freezing on the viability of cryopreserved cultured epithelial grafts. Implants were prepared at room temperature with 10% glycerol and 15% dextran (70k
The cryopreservation medium containing d) was equilibrated for 30, 60, and 120 minutes. After thawing, the cells of the transplant were assayed for R, CFE, and CFC.
The results were expressed as a percentage of the control graft.

FIG. 5 is a bar graph showing the effect of storage temperature on the viability of cryopreserved cultured epithelial grafts. Graft 10% at room temperature
Equilibrate for 30 minutes in cryopreservation medium containing 15% dextran in glycerol, freeze at -85 ° C, and store in liquid nitrogen vapor at -180 ° C, or -80 ° C in a mechanical freezer, or Carbon dioxide (dry ice, sublimation point -77
° C) and stored at -65 ° C. After thawing, the R, CFE and CFC of the graft were measured. The results were expressed as percent of unfrozen, unpreserved transplants as control grafts.

FIG. 6 is a bar graph showing the effect of the cryopreserved cultured epithelial graft after thawing. The grafts were equilibrated for 60 minutes in cryopreservation medium before freezing. After cryopreservation at -180 ° C for 1-3 days, the explants were rapidly thawed. At 0, 30, and 60 minutes after melting, the sample was removed from the CPM and rinsed with Lactated Ringer's solution for 1 to 2 hours. Measure cell recovery, CFE, CFC of the graft after treatment,
Control grafts were expressed as a percentage of unfrozen, non-preserved grafts.

DETAILED DESCRIPTION Cultured human epithelial cell sheets can serve as permanent autografts for the treatment of skin wounds. As a temporary allograft material, this sheet can be used as a treatment for chronic skin ulcers and resected graft donors, and also as a highly effective burn dressing it can. Sheets can be prepared with a culture system developed by Reinwald and Green. In this system, continuously cultured epithelial cells rapidly divide on the surface of a tissue culture dish or flask, eventually forming a loose, layered, polarized sheet of cells that are tightly bound to one another. A micrograph of a cross section of the culture sheet is shown in FIG. The stratified epithelial cell culture is treated with an enzyme such as despase, peeled off as a binding cell sheet, fixed to a non-adherent material such as vaseline or gauze impregnated with vaseline, and fixed in the operating room. And transport it to the patient until treatment.

Significant limitations in the use of cultured epithelial cell grafts are their extreme fragility and short shelf life. If the graft has been away from its culture support for more than 8 hours,
Measuring the ability of the isolated cells to recover growth and colony formation under optimal cell conditions has shown experimental results that the cell viability of the graft is substantially reduced. For this reason, sales of cultured epithelial cell sheets have been limited locally. That is, in order to start the separation in the culture facility,
Add despase to the culture first, then add at least 8
The hospital had to be close enough to the production center to prepare, transport and administer the graft within hours. The actual time of transfer was only 2.3 hours due to the time required to prepare the implant. Operating room schedules and graft arrival times had to be carefully coordinated. Preservation of the sheets was not possible at any time, which made it impossible to keep inventory of timed products.

Observations will clearly explain why the viability of the detached epithelial cell sheets is such a short time. In other words, epithelial cells lose potential for division when kept temporarily under conditions that prevent them from recombining to the surface as single cells separated from cultures by treatment with trypsin and EDTA, and eventually lose their potential for division. It was found to undergo differentiation.

Experiments measuring viability over 8 hours revealed the following. That is, the temperature at which the graft treated with the despase was maintained was determined by the colony forming ability (CFE) and the total cell number of the recovered colony forming cells (CFC or recovered whole cells × CF).
The temperature was very critical for cell viability as measured by E). When the cultured epithelial sheet is stored at a temperature equal to or slightly lower than the physiological temperature condition, CFE of fresh tissue cannot be maintained. Similarly, careful control of media conditions, pH, carbon dioxide / oxygen balance, etc., did not apparently give any effect at 4 ° C. If the culture sheet is maintained at the limit temperature range of 10 ° C. to 25 ° C., preferably 13 ° C. to 23 ° C., the CFE can be maintained for about 20-30 hours or longer. However, for now, retaining its usefulness as a wound dressing,
There is no way to store culture sheets for more than one day. A freshly prepared product for wound healing applications cannot be preserved as a preservative.

Looking at the history of cryopreservation methods, optimizing a protocol for cryopreserving specific cells involves using other types of cells, using the same type of cells from different species, or using other cells in the tissue. In some cases, it is understood that good results are not necessarily obtained. The method of freezing the suspension of keratinocytes, when applied to a complete sheet,
Only unsatisfactory results have been obtained. Freezing whole tissue for actual transplantation was not successful with any tissue type to our knowledge (Canc
See EP 0296475 by eda et al.).

The method disclosed herein for cryopreserving a confluent sheet of living cultured epithelial cells after separation from the culture substrate can be used to maintain mitotic or cell-forming ability at acceptable levels or to maintain sheet integrity. Hold. In summary,
The method comprises four steps. First, the cell sheet
Equilibrate the cryoprotectant solution for a time sufficient to thoroughly mix or replace the intracellular and intercellular water with the cryoprotectant. Second, the sheet cools the cryoprotected cells at a sufficiently slow rate of temperature drop, preferably from about -180 ° C to -196 ° C, to avoid ice crystal formation and subsequent damage. Frozen sheets can be stored at about -180 ° C for long periods and at higher temperatures for short periods, for example at -65 ° C. Before use, the sheet should be air or other gas
Warm up to room temperature within -3 minutes, then warm quickly, for example in a water bath, to completely melt. Fourth,
The cryoprotectant is removed by rinsing the cultured epithelial cell sheet with an isotonic buffer such as Ringer's lactate solution.

 The detailed steps are disclosed below.

Preparation of Cultured Epithelial Cell Sheet Epithelial cells (keratinocytes) are seeded in T150 culture flasks (Costar) at a specific gravity that reaches confluence in 10-12 days. Cultures of frozen cell suspensions prepared from several epithelial cell lines can be used for allogeneic transplantation. Cells obtained by biopsy from burn patients can be used for autologous transplantation. Culture is performed at 37 ° C using a gas-tight flask.
FAD medium (1 part Hams F12 medium supplemented with adenine, 3 parts Dulbecco's modified Eagle medium (DME), 10% fetal bovine serum (FB
S), 0.4 μg / ml hydrocortisone, 1 × 10 ⁻¹⁰ M cholera toxin, 2 × 10 ⁻⁹ M triiodothyronine). They are further grown in the presence of lethally radiated 3T3 fibroblasts. See U.S. Pat. No. 4,016,036. 10 ng / ml epidermal growth factor is included in the initial inoculation.

When the cell culture reaches confluence, the cell culture is used for preparing an explant. The supernatant medium is aspirated, 40 ml of despase II (Boehringer Mannheim) is added to the flask to a final concentration of 2.5 mg / ml (about 1.2 U / ml) and incubated at 37 ° C. Once the edge of the sheet has peeled off (45 minutes), bake off the top of the flask with a soldering iron.

 The enzyme solution is replaced with 20 ml of DME medium. Then the epithelium
The cell sheet is rinsed once more with 20 ml of DME medium. Second rinse
The remaining solution is aspirated, leaving 3-4 ml of the solution, and the solution is aspirated.
e 5 × 10cm gauze impregnated with cheese)
 Pons) overlaps the cells on the surface facing the gauze bandage
On the cell sheet that has been peeled off. Then join
Staple (RigaClip d)
Chikon / J & J) to bandage. Cut the sheet wide
Cut, fixed, and transplanted with epithelial cell
Transfer to 0 mm dish. To prevent the end of the graft from lifting
Use a rubber policeman to hold it down. DME12m
l slowly and then transfer the dishes to the storage container.
You. (Fig. 1 shows a cross section of a typical culture sheet by this method.
FIG. ) Cell recovery (R) and colony shape
Capacitance analysis (CFE) is a mitotic total cell count (CFC)
Do to make sure. In this operation,
Optimal conditions that protect the viability during the process are determined. That
The conditions are the composition of the cryoprotective medium and the cryoprotective medium prior to freezing.
Local equilibration time, freezing rate, storage temperature, thawing process, rinsing
This is about the refining process and the subsequent storage.

The colony forming ability (CFE) analysis is performed on transplants prepared as in the examples shown below after storing for 1 to 3 days after freezing. The unfrozen graft is analyzed as a control. The fixing clip is removed using forceps and the ready cell sheet is separated into a single cell suspension of isotonic buffer containing trypsin (0.05%) and EDTA (0.01%). The enzymatic reaction was stopped by the addition of bovine serum, followed by a 10-fold dilution by adding 0.5 ml of cell suspension to 4.5 ml of FAD,
This is performed twice in succession. An aliquot of the first cell suspension is counted using a hemocytometer. Final cell density of 1000-2000
/ ml of cell suspension, 1 ml of cell suspension
Is seeded in a 100 mm dish containing 12 ml of FAD medium containing 3T3 cells killed by irradiation.

After 10-14 days, the cultured cells are fixed with 10% formalin in phosphate buffered saline and stained with a mixed solution of 1% rhodamine and 1% Nile Blue A. Colonies are counted using a dissecting microscope and scored as growing or stunted. CFE is calculated as follows.

The percent recovery and percent CFE are calculated as follows:

Here, FZ means frozen and FS means fresh.

Total recovery of colony forming cells (CFC) is calculated as follows.

CFC = percentage of total recovered cells x percentage of recovered colony forming ability (CFE) The CFE% using the protocol of the present invention is usually 3%.
Over 5%, typically over 40%, often 50
% Or more.

Cryopreservation protocols The development of cryopreservation methods requires the accumulation of independent studies, stitching together optimal elements, and optimizing each element of the process to identify the final step . Each optimal freezing, storage, thawing, and rinsing step that is compatible with maintaining maximum viability needs to be specified. These elements are identified by colony forming ability analysis as described above.

Standard cryopreservation media include bovine serum, glycerol,
It consists of a physiologically balanced salt solution to which a cell-permeable glass former has been added (for example, a cell culture medium). Cryopreservation of cells in suspension is successful, but the ability to preserve the viability of cells removed from the cell sheet is not well understood. FIG. 2 shows the beneficial effects of supplementing a standard cryopreservation medium with additional components, a non-cell permeable glass former (such as dextran). It should be noted that using a non-cell penetrant with a cell penetrant significantly improves cell recovery and thus viability (CFC) compared to using the cell penetrant alone. Must.

FIG. 3 shows that the use of a non-cell permeable glass former resulted in improved viability. These drugs have a complex carbohydrate polymer structure. The non-cell penetrating glass former preferably comprises approximately 50-500 kilodaltons (kd) of high molecular weight dextran, preferably 50-70 kd of chondroitin sulfate, polvinyl vinyl pyrrolidone, polyethylene glycol, a mutated starch such as hydroxyethyl starch. I can give it. Preferably, the cell penetrating glass former is glycerol, but propylene glycol, ethylene glycol, dimethyl sulfoxide, and other penetrating glass formers known from the prior art can also be used.

At the beginning of the cryopreservation step, it is necessary to soak the cell sheet for freezing in the cryoprotective medium for a sufficient time to equilibrate the cells with the cryoprotectant. FIG.
Shows the effect on viability when equilibrating the cultured epithelial sheet against the cryoprotectant for a long time before freezing. The data shows that the sheet can be equilibrated to the cryoprotectant for up to 2 hours before freezing without affecting the viability of the cells in the cryopreserved sheet. Equilibration is
Typical conditions are 17 ° C to 25 ° C for approximately 30-60 minutes.
And usually in a cryoprotective solution in a thin storage dish at room temperature.

Following equilibration, the sheet and the dish containing the cryoprotectant solution are
Seal to prevent gas and water from permeating. The sheet in the sealed container is cooled to at least -65 ° C (using dry ice), preferably to -120 ° C, and to about -180 ° C to -196 ° C for longer storage. The cooling rate is slow (0 ° C / min or less) from 0 ° C to -80 ° C. This helps to suppress the formation of ice crystals. Preferably, the cooling is at a temperature of -40 ° C to -100 ° C, preferably at a temperature of -80 ° C to -85 ° C, with a cooling ratio adjusting cooling effect such as a commercially available program cell freezer (Climond No. 1010/2700). Do first by It is then transferred to a liquid nitrogen storage container and maintained in liquid nitrogen vapor to further reduce its temperature.

In a preferred freezing protocol, the tissue is approximately 4 ° C.
Cool the sheet in the sealed container until The sheet is then cooled at 1 ° C. per minute. After the temperature of the sheet has been reduced to at least -65 ° C, preferably to -85 ° C, the container is transferred to a liquid nitrogen freezer and is about -180 ° C (nitrogen vapor) or -196 ° C (liquid nitrogen).
To save.

Storage of the tissue at -180 ° C or below can maintain the colony forming ability of the cells over storing the sheet at higher temperatures, as shown in FIG. Storage at -180 ° C using liquid nitrogen vapor is better than storing at -80 ° C using a mechanical freezer or -77 ° C using dry ice. Is evident from the data. Storage at -80 ° C or -77 ° C dramatically reduces viability during the first two or three days. In contrast, viability at -180 ° C is more stable for longer periods. Implants are preferably shipped at -180 ° C.

To thaw the sheet, remove the sealed container from the liquid nitrogen freezer and hold in air at room temperature and for 1 minute to approximately 3-5 minutes. This is from 20 ° C / min to 100
Produces a heating rate between ° C / min. The implant is then warmed to room temperature regardless of the warming rate. Preferably,
In the final step, the sealed container is submerged in a water bath until the graft has melted. Thereby, the frozen sheet does not crack. Melting can be performed by immersing in a water bath at 37 ° C. for 1.25 minutes. If the temperature of the water bath is 25 ° C., melting will take approximately 1.5 minutes. The water bath can be omitted and the sheet can be melted at room temperature instead. However, this requires 27 minutes and often reduces cell viability.

The thawed sheet is removed from the cryoprotectant within one hour, preferably as soon as possible. Once the sheet is thawed, open the container and replace the cryopreservation solution with an isotonic buffer of physiological pH (about 6.8-7.4), preferably FAD medium or Ringer's lactate, to dilute the cryoprotectant. Table 1A shows that not all isotonic buffers at physiological pH help to dilute the cryoprotectant. Phosphate buffered saline and standard saline obviously reduce viability as determined by CFE. The molten sheet is preferably
Equilibrate with buffer for 15 minutes. In addition, the CFE can be slowly lowered and left to stand for up to 4 hours. (Table 5) The explants were removed from the cryoprotective medium and placed in a rinse solution for 15 minutes at room temperature. Perform colony forming ability analysis,
CFE recovery (rCFE) was calculated as% of unfrozen, non-preserved control graft. FAD; F12 / DME keratinocyte growth medium, PBS; phosphate buffered saline, normal saline; 0.9% saline The explants were removed from the cryoprotective medium, placed in a FAD rinse solution and allowed to stand at room temperature for the time indicated above. A colony forming assay was performed and CFE recovery (rCFE) was calculated as% of unfrozen, non-preserved control explants.

Those that limit the final step of cryopreservation were tested and are shown in FIG. For production of a viable and fully restored cell sheet with normal metabolic function, the cultured epithelial sheet is equilibrated in the cryoprotectant for up to 1 hour before freezing, and freezing, thawing, and leaving in the cryoprotectant is 1 hour. The data shows that over time, the final rinsing in Ringer's lactate solution is up to 2 hours.

Sheets prepared as disclosed above can be used to surgically cover clean skin wounds, such as burns and ulcers, using a gauze backing as a bandage with a growing layer that contacts the surface of the wound. . The allograft sheet actually falls off, but helps safe and effective wound protection before rejection. Autograft sheets are generally permanent and differentiate until they form completely layered skin. The high CFC and CFE values performed by this cryopreservation method indicate that the majority of the individual cells in the sheet are mitotic and many epithelial cell colonies are formed in the wound.

 The present invention is further described by the following non-limiting examples.

Example 1 15% dextran (70,000 mw)
And 10% glycerol in a shallow storage dish containing a culture solution containing a cryoprotectant. The container should be sealed to prevent gas and water from permeating, and should be well-filled with solution so that little space remains, and the implant should be kept at room temperature.
Equilibrate for 30 minutes.

The sheet is cooled in a sealed container until the piece of tissue is about 4 ° C. The sheet is then approximately -85 ° C
Cool 1 ° C per minute until When the sheet temperature reaches -85 ° C, move the container to a liquid nitrogen freezer,
Store in liquid nitrogen vapor (about -180 ° C).

To melt the sheet, remove the sealed container from the liquid nitrogen freezer and leave it at room temperature in air for approximately one minute. It is then placed in a 37 ° C. water bath for about 75 seconds to thaw. After thawing, open the container and place the sheet in Ringer's lactate solution at physiological pH for 5 minutes. The buffer is then changed and the sheet is allowed to equilibrate in the medium for at least 10 minutes.

Example 2 The implant was 15% hydroxyethyl starch (Hespan )
And 10% glycerol in a cryoprotective solution
Equilibrate at room temperature for 15 minutes in a fresh storage dish. After equilibration,
Containers are sealed to prevent gas and water from permeating
You.

The sheet in the sealed container has a tissue of about 4 ° C
Cool down to Following this step, the sheet is cooled to approximately -85 ° C at 1 ° C per minute. When the sheet reaches -85 ° C, transfer to a liquid nitrogen freezer and store in liquid nitrogen (about -196 ° C).

To melt the sheet, remove the sealed container from the liquid nitrogen freezer and leave it at room temperature in air for approximately one minute. Then place in a water bath and allow to thaw. After thawing, open the container and set the sheet to physiological pH lactated Ringer's solution or serum-free DME / F1
2 Soak in keratinocyte growth medium for 5 minutes. After 5 minutes, the buffer is changed and the sheets are allowed to equilibrate in the medium for 4 hours.

The invention can be implemented in other specific forms. Other implementations are within the following claims.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Sharmer, Alexander United States 02178 Massachusetts, Belmont, Frederick Street 15 (72) Inventor Odyssey, Richard United States 02162 Massachusetts, Newton Lower Falls, Klehoe Drive 15 ( 56) References JP-A-2-174848 (JP, A) JP-A-59-21621 (JP, A) JP-A-56-29992 (JP, A) JP-A-2-500800 (JP, A) Cryobiology, Vol. 5 (1985), p. 495-498 Cryobiology, Vol. 5 (1972), p. 441-449

Claims (27)

(57) [Claims] 1. An improved method for maintaining a cryopreserved viable epithelial cell binding sheet comprising the following steps. A. providing a cultured and layered multi-layered epithelial cell sheet separated from its culture substrate; B. a cryoprotective solution comprising a non-cell permeable glass former combined with a cell permeable glass former C. The temperature of the sheet in the solution for cryoprotection is −65 ° C. or less and −196 ° C.
After cooling as described above, D. The sheet obtained in the operation of Step C is −65 ° C. or less and −196 ° C.
E. Thaw the cryopreserved sheet to prepare a sheet of viable cells with the ability to retain stratified mitosis, storing at a temperature above. Step that the sheet is useful as a skin wound dressing. 2. The method according to claim 1, wherein the cryoprotective solution comprises from 5% to 20% of said non-cell penetrating glass former. 3. The method of claim 1, wherein the non-cell penetrating glass forming agent is selected from the group consisting of dextran and its derivatives, polyethylene glycol, polyvinylpyrrolidone, hydroxyethyl starch, chondroitin sulfate, and mixtures thereof. Method. 4. The method according to claim 1, wherein the non-cell permeable glass forming agent is dextran or hydroxyethyl starch. 5. The method of claim 4 wherein the glass former is dextran having a molecular weight of about 70 to 500 kilodaltons. 6. The cryoprotective solution according to claim 1, wherein the cryoprotective solution contains 10% by weight of a cell-permeable glass-forming agent selected from the group consisting of glycerol, dimethyl sulfoxide and a mixture thereof. Method. 7. The method according to claim 6, wherein the cell-permeable glass forming agent is glycerol. 8. The method of claim 1, wherein the immersion step is performed for 15 to 180 minutes. 9. The method according to claim 9, wherein the sheet is immersed in the cryoprotective solution at 17 ° C.-30 ° C. for 120 minutes, and then cooled to 4 ° C. within 60 minutes. 10. The method of claim 1 wherein the step of cooling is performed at a rate of 1 ° C. per minute to cool the sheet from 4 ° C. to below −80 ° C. 11. The method of claim 1 wherein the sheet is cooled slowly to -80.degree. C. and then to a temperature in the range of -180.degree. 12. The method according to claim 1, wherein the step of cooling and storing is at or below -120 ° C
The method according to claim 1, which is performed at 96 ° C or higher. 13. The method of claim 1 wherein the steps of cooling and storing are performed in a rigid, transparent, sterile, gas impermeable container. 14. The method of claim 1 wherein the steps of cooling and storing are performed at a temperature below -180.degree. C. and above 196.degree. 15. Cooling the sheet at a temperature below -180 ° C and above -196 ° C and allowing the sheet to thaw to a temperature range from -120 ° C to -80 ° C is accomplished by incubating the sheet in a gas. The method of claim 1, wherein the subsequent thawing is performed by immersion in a solution. 16. The method of claim 1, wherein the thawing step is performed for 1 to 5 minutes.
The method of claim 1 comprising heating the sheet once to -80 ° C and then raising it to room temperature. 17. The method according to claim 1, wherein the following steps are added. F. Remove cryoprotectants from thawed sheets by transferring to prepared salt solution for up to 4 hours to maintain the viability of the sheets. 18. The prepared salt solution may be a culture solution such as DME / F12 or a lactated Ringer's solution.
18. The method of claim 17, wherein the method is on). 19. The method of claim 1, wherein step C is performed by exposing the sheet to liquid nitrogen vapor. 20. The method according to claim 1, wherein the epithelial cells are keratinocytes. 21. The method according to claim 1, wherein the retained sheet of viable cells is characterized by a colony forming ability of at least 35%. 22. The method of claim 1, wherein the retained sheet of viable cells is characterized by a cell recovery of at least 40%. 23. The method of claim 1 including the additional step of placing the sheet on a support prior to step C. 24. A method for applying as a skin wound dressing to a patient, wherein when thawing a sheet at a temperature of -180 ° C. or less, the temperature of the sheet is increased from 20 ° C. per minute to 100 ° C. per minute. In the ratio between -120 ℃ to -80 ℃ increase,
2. The method according to claim 1, further comprising the step of raising the temperature to between 20 ° C and 37 ° C. 25. Exposing the sheet to a gas at -80 ° C.
25. The method of claim 24, wherein the temperature is increased to a temperature between 20 ° C and 37 ° C in the solution. 26. The method of claim 24, further comprising the step of incubating the sheet in a conditioned salt solution for up to 4 hours to remove the cryoprotectant bathing the sheet. 27. The method according to claim 5, wherein the dextran is combined with a cell permeable agent, glycerol.